The overall aim of this application is to develop methods to identify, quantify, and isolate radioresistant and possibly chemoresistant hypoxic cells from solid tumors. Factors which might influence the nature and development of tumor hypoxia will also be examined. The results obtained in the previous grant period indicate that chronically and acutely hypoxic cells in tumors may be identified using fluorescent molecules with limited ability to diffuse through solid tissues (Hoechst 33342, DiOC7) fluorescent molecules which are metabolized and bound by hypoxic cells (AF-2, NFVO),and fluoroescent molecules whose metabolism is dependent upon the redox state of the cell (hydroethidine). In the next grant period, the investigators will 1) continue to search for new agents less toxic, more stable and/or more fluorescent than those currently available, 2) determine whether combining fluorescent redox probes, perfusion probes and hypoxia probes can improve their ability to identify and sort hypoxic tumor cells, 3) examine the relationship between tumor hypoxia, tumor respiration rate and vascular density, 4) determine whether hypoxic tumor cells adapt to low oxygen conditions and may therefore be susceptible to oxygen toxicity (reperfusion injury) and 5) examine characteristics of normal cells in tumors which might influence tumor hypoxia. Multicell spheroids composed of Chinese hamster V79 lung fibroblasts and WIDR human adenocarcinoma cells will be used to screen fluorescent compounds for use as hypoxia markers. The SCCVII squamous cell carcinoma growing in C3H mice will be used to test these probes for their ability to identify hypoxic cells in vivo. Human tumor xenografts growing in nude mice will be used to address the question of the role of tumor respiration rate and vascular density in development of tumor hypoxia. Fluorescence activated cell sorting and fluorescence imaging methods will be applied to identify and select cells for clonogenicity measurement following radiation treatment. Results from these studies should significantly improve our understanding of solid tumor biology and may ultimately provide us with methods to identify and quantify hypoxic cells in solid tumors.